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Quantify DNA in gel electrophoresis bands

gel electrophoresis quantify

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7 replies to this topic

#1 Sylvia Joanne

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Posted 26 March 2013 - 06:24 PM

Hye,

I have been doing amplification of a particular GOI in PCR and running gel electrophoresis to determine the successful amplification. Now, I am interested to determine the amount of DNA present in each of my band. My gel of each samples usually gives me two bands. I want to be able to quantify each of the bands and compare it with intensity of other sample bands.

Anyone can give me idea on how I can quantify my gel bands please?

Thank you

#2 jerryshelly1

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Posted 26 March 2013 - 06:31 PM

DNA ladder can provide a good estimate on the amount of DNA that is present in your bands. Look at the DNA ladder image on the manufacturer's website. It will give you a picture with the sizing of your ladder and also the ng of DNA at each sized band.

If you have HindIII DNA ladder, I would use that. I prefer it to semi-quantify the amount of DNA at a particular size.

#3 pcrman

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Posted 26 March 2013 - 11:04 PM

I believe Sylvia wants to quantify the amount of the DNA not the size. Yes you can, and can do so in many ways. It is called DNA Quantification by Gel Densitometry. The commonly used program is ImageJ, a free program. You can use other programs such as Photoshop to obtain the mean pixel intensity value by using the histogram tool

#4 jerryshelly1

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Posted 27 March 2013 - 07:18 AM

You can quantify the amount of DNA based on the DNA ladder you are using. http://www.norgenbio...oduct.php?ID=50

Every new DNA ladder provides you with both the size and the relative concentration of the DNA ladder band, at that particular size. This is the method I use to determine the vector:insert ratio for my ligation experiments. Remember, this is semi-quantitative, but it provides a decent estimate. PCRman is correct. Using a visualization software would make your readings more accurate, but judging by your eye is also a good representation.

#5 Sylvia Joanne

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Posted 27 March 2013 - 07:05 PM

Thank you for the replies. I downloaded and tried the ImageJ but the peaks I get after analyzing my bands is not right. Eg: I have 2 bands, but it gives me half peak, one full peak n another half peak. Therefore I cant analyse the peaks and get the relative densities. Any other free software suggestions?
Thank you.

#6 jerryshelly1

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Posted 28 March 2013 - 04:22 AM

I'm sure you can get it to work. Open up the provided gel image in ImageJ and try to obtain the same numbers the have on there NIH page. http://rsbweb.nih.gov/ij/docs/

You probably need to adjust your scales. Good luck

#7 Ubiquitous

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Posted 28 March 2013 - 01:16 PM

Does densiometry for DNA have similar pitfalls as it does for protein WBs?

http://www.ncbi.nlm....pubmed/19517440
http://onlinelibrary...800720/suppinfo

Edited by Ubiquitous, 28 March 2013 - 01:18 PM.


#8 Sylvia Joanne

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Posted 28 March 2013 - 09:01 PM

Hye,
Thanx. I managed to get the Image J to work. Because it is gray scaled, the peaks were upside down actually. There is a program from Thermo Scientific MyImageAnalysis also very good.
Thanx for all the help..





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